TY - JOUR
T1 - Patched 1 regulates Smoothened by controlling sterol binding to its extracellular cysteine-rich domain
AU - Kinnebrew, Maia
AU - Woolley, Rachel E.
AU - Ansell, T. Bertie
AU - Byrne, Eamon F.X.
AU - Frigui, Sara
AU - Luchetti, Giovanni
AU - Sircar, Ria
AU - Nachtergaele, Sigrid
AU - Mydock-McGrane, Laurel
AU - Krishnan, Kathiresan
AU - Newstead, Simon
AU - Sansom, Mark S.P.
AU - Covey, Douglas F.
AU - Siebold, Christian
AU - Rohatgi, Rajat
N1 - Funding Information:
We thank J. Ferrell and D. Herschlag for discussions, the staff of beamline I24 (MX14744 and MX19946) at the DLS, UK for assistance, and J. S. Tamaresis (Department of Biomedical Data Science, Stanford University School of Medicine) for expert advice on statistical analysis. C.S. was supported by grants from Cancer Research UK (C20724/ A26752) and the European Research Council (647278), R.R. by grants from the National Institutes of Health (GM118082 and GM106078), D.F.C. by a grant from the NIH (HL067773), M.S.P.S. by grants from Wellcome (208361/Z/17/Z) and BBSRC (BB/R00126X/1), M.K. by a predoctoral fellowship from the National Science Foundation, R.E.W. and T.B.A. by Wellcome Trust DPhil studentships (203726/Z/16/Z and 102164/Z/13/Z), and G.L. by a predoctoral fellowship from the Ford Foundation. Additional support was provided by the Wellcome Trust Core Award (203141/Z/16/Z).
Publisher Copyright:
Copyright © 2022 The Authors,
PY - 2022/6
Y1 - 2022/6
N2 - Smoothened (SMO) transduces the Hedgehog (Hh) signal across the plasma membrane in response to accessible cholesterol. Cholesterol binds SMO at two sites: one in the extracellular cysteine-rich domain (CRD) and a second in the transmembrane domain (TMD). How these two sterol-binding sites mediate SMO activation in response to the ligand Sonic Hedgehog (SHH) remains unknown. We find that mutations in the CRD (but not the TMD) reduce the fold increase in SMO activity triggered by SHH. SHH also promotes the photocrosslinking of a sterol analog to the CRD in intact cells. In contrast, sterol binding to the TMD site boosts SMO activity regardless of SHH exposure. Mutational and computational analyses show that these sites are in allosteric communication despite being 45 angstroms apart. Hence, sterols function as both SHH-regulated orthosteric ligands at the CRD and allosteric ligands at the TMD to regulate SMO activity and Hh signaling.
AB - Smoothened (SMO) transduces the Hedgehog (Hh) signal across the plasma membrane in response to accessible cholesterol. Cholesterol binds SMO at two sites: one in the extracellular cysteine-rich domain (CRD) and a second in the transmembrane domain (TMD). How these two sterol-binding sites mediate SMO activation in response to the ligand Sonic Hedgehog (SHH) remains unknown. We find that mutations in the CRD (but not the TMD) reduce the fold increase in SMO activity triggered by SHH. SHH also promotes the photocrosslinking of a sterol analog to the CRD in intact cells. In contrast, sterol binding to the TMD site boosts SMO activity regardless of SHH exposure. Mutational and computational analyses show that these sites are in allosteric communication despite being 45 angstroms apart. Hence, sterols function as both SHH-regulated orthosteric ligands at the CRD and allosteric ligands at the TMD to regulate SMO activity and Hh signaling.
UR - http://www.scopus.com/inward/record.url?scp=85131702385&partnerID=8YFLogxK
U2 - 10.1126/sciadv.abm5563
DO - 10.1126/sciadv.abm5563
M3 - Article
C2 - 35658032
AN - SCOPUS:85131702385
SN - 2375-2548
VL - 8
JO - Science Advances
JF - Science Advances
IS - 22
M1 - eabm5563
ER -